[1]柏梦焱,袁珏慧,孙嘉丰,等.基于CRISPR-Cas9基因编辑技术创制大豆gmnark超结瘤突变体[J].大豆科学,2019,38(04):525-532.[doi:10.11861/j.issn.1000-9841.2019.04.0525]
　BAI Meng-yan,YUAN Jue-hui,SUN Jia-feng,et al.Generation ofgmnarkMutant with SupernodulationviaCRISPR-Cas9 in Soybean[J].Soybean Science,2019,38(04):525-532.[doi:10.11861/j.issn.1000-9841.2019.04.0525]
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基于CRISPR-Cas9基因编辑技术创制大豆gmnark超结瘤突变体

《大豆科学》[ISSN:1000-9841/CN:23-1227/S] 卷: 第38卷 期数: 2019年04期 页码: 525-532 栏目: 出版日期: 2019-07-20
Title:
Generation ofgmnarkMutant with SupernodulationviaCRISPR-Cas9 in Soybean
作者:
柏梦焱1; 2; 袁珏慧1; 2; 孙嘉丰1; 2; 厉苏宁2; 3; 关跃峰2
(1.福建农林大学 资源与环境学院，福建 福州 350002； 2.福建农林大学 海峡联合研究院园艺植物生物学与代谢组学研究中心，福建 福州 350002； 3.福建农林大学 生物科学学院，福建 福州 350002)
Author(s):
BAI Meng-yan1; 2;  YUAN Jue-hui1; 2;  SUN Jia-feng1; 2;  LI Su-ning2; 3;  GUAN Yue-feng2
(1.College of Resources and Environment, Fujian Agriculture and Forestry University, Fuzhou 350002, China; 2.FAFU-UCR Joint Center for Horticultural Biology and Metabolomics, Haixia Institute of Science and Technology, Fujian Agriculture and Forestry University, Fuzhou 350002, China; 3.College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China)
关键词:
大豆; 基因编辑; 结瘤自我调节机制; 移码突变
Keywords:
Soybean;  Genome editing;  Autoregulation of nodulation;  Frameshift mutation
DOI:
10.11861/j.issn.1000-9841.2019.04.0525
文献标志码:
A
摘要:
为研究豆科作物结瘤自我调节机制(autoregulation of nodulation, AON)的作用机理，运用CRISPR-Cas9基因编辑技术创制大豆品种华春6号超结瘤gmnark突变体，设计3条靶向目的基因GmNARK的特异sgRNA，构建CRISPR-Cas9敲除载体。通过毛根转化试验选取sgRNA-B和sgRNA-C两条编辑效率较高的sgRNA用于大豆稳定转化，在T1代筛选出8种不同突变类型的突变体，在T2代通过营养液水培试验证明其中5种突变体有超结瘤的表型。选取gmnark-A突变体作进一步表型分析，发现其具有超结瘤、植株矮小和叶片深绿的表型。本研究所创制的gmnark突变体是研究AON途径作用机制与根瘤发育的重要遗传材料，同时此新种质资源具有作为“绿肥”与其它作物进行间作或轮作的巨大潜力。
Abstract:
In order to study the mechanism of the autoregulation of nodulation (AON) in the legumes, the gene editing technology of CRISPR-Cas9 system was used to create thegmnarkmutant of the soybean variety Huachun 6, and three specific sgRNAs were designed to locate the targeted geneGmNARKand constructed into a CRISPR-Cas9 knockout vector. Two sgRNAs with high editing efficiency, sgRNA-B and sgRNA-C, were selected for stable transformation through hairy root transformation experiments. Furthermore, eight mutants of different mutation types were screened in T1 generation and five of them had the phenotype of supernodulation which is verified by hydroponic experiment. In the further phenotypic analysis ofgmnark-Amutant, it was found thatgmnark-Aowned a larger number of nodules, smaller shoot and root and greener leaves than WT. These data proved that thegmnarkmutant created in the study is an important genetic material for studying the mechanism of AON pathway and the development of nodule. At the same time, this new germplasm resource has great potential in intercropping with other crops as ‘green manure’.

参考文献/References:

［1］Collier R, Tegeder M. Soybean ureide transporters play a critical role in nodule development, function and nitrogen export［J］. The Plant Journal, 2012, 72（3）: 355-367.

［2］Herridge D F, Peoples M B, Boddey R M. Global inputs of biological nitrogen fixation in agricultural systems［J］. Plant Soil, 2008, 311（1-2）: 1-18.
［3］Oldroyd G E, Downie J A. Coordinating nodule morphogenesis with rhizobial infection in legumes［J］. Annual Review of Plant Biology, 2008, 59: 519-546.
［4］Oldroyd G E, Murray J D, Poole P S, et al. The rules of engagement in the legume-rhizobial symbiosis［J］. Annual Review of Genetics, 2012, 45: 119-144.
［5］Brett J F, Arief I, Satomi H, et al. Molecular analysis of legume nodule development and autoregulation［J］. Journal of Integrative Plant Biology, 2010, 52 (1): 61-76.
［6］Okamoto S, Shinohara H, Mori T, et al. Root-derived CLE glycopeptides control nodulation by direct binding to HAR1 receptor kinase［J］. Nature Communication, 2013, 4: 2191.
［7］Reid D E, Ferguson B J, Gresshoff P M, et al. Inoculation- and nitrate-induced CLE peptides of soybean control NARK-dependent nodule formation［J］. Molecular Plant-Microbe Interactions, 2013, 24（5）: 606-618.
［8］Searle I R, Men A E, Laniya T S, et al. Long-distance signaling in nodulation directed by a CLAVATA1-like receptor kinase［J］. Science, 2003, 299(5603): 109-112.
［9］Sasaki T, Suzaki T, Soyano T, et al. Shoot- derived cytokinins systemically regulate root nodulation［J］. Nature Communication, 2014, 5: 4983.
［10］Daniela T, Zhe Y, Dennis B, et al. Systemic control of legume susceptibility to rhizobial infection by a mobile microRNA［J］. Science, 2018, 362（6411）: 233-236.
［11］Takahara M, Magori M, Soyano T, et al. Too much love, a novel Kelch repeat-containing F-box protein, functions in the long- distance regulation of the legume-Rhizobiumsymbiosis［J］. Plant Cell Physiology, 2013, 54（4）: 433-447.
［12］Ogawa M, Shinohara H, Sakagai Y, et al.ArabidopsisCLV3 peptide directly binds CLV1 ectodomain［J］. Science, 2008, 319（5861）: 294.
［13］Ran F A, Hsu P D, Wright J, et al. Genome engineering using the CRISPR/Cas9 system［J］. Nature Protocols, 2013, 8（11）: 2281-2308.
［14］Feng Z, Zhang B, Ding W, et al. Efficient genome editing in plants using a CRISPR/Cas system［J］. Cell Research, 2013, 23(10): 1229-1232.
［15］Ma X L, Zhang Q, Zhu Q, et al. A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicotplants［J］. Molecular Plant, 2015, 8(8): 1274-1284.
［16］Burgess. Technology: A CRISPR/Cas9 genome editing tool［J］. Nature Reviews Genetics, 2013, 14（2）: 80.
［17］Fu Y, Foden J A, Khayter C, et al. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells［J］. Nature Biotechnology, 2013, 31(9): 822-826.
［18］Miao J, Guo D, Zhang J, et al. Targeted mutagenesis in rice using CRISPR-Cas system［J］. Cell Research, 2013, 23(10): 1233-1236.
［19］Duan J, Lu G, Xie Z, et al. Genome-wide identification of CRISPR/Cas9 off-targets in human genome［J］. Cell Research, 2014, 24(8): 1009-1012.
［20］Liang Z, Zhang K, Chen K, et al. Targeted mutagenesis inZea maysusing TA1LENs and the CRISPR［J］. Journal of Genetics and Genomics, 2014, 41(2): 63-68.
［21］Masafumi M, Toki S, Endo M. Parameters affecting frequency of CRISPR/Cas9 mediated targeted mutagenesis in rice［J］. Plant Cell Reports, 2015, 34(10): 1807-1815.
［22］Sun Y, Zhang X, Wu C, et al. Engineering herbicide-resistant rice plants through CRISPR/Cas9-rediated homologous recombination of acetolactate synthase［J］. Molecular Plant, 2016, 9(4): 628-631.
［23］Jacobs T B, LaFayette P R, Schmitz R J, et al. Targeted genome modifications in soybean with CRISPR/Cas9［J］. BMC Biotechnology, 2015, 15（1）: 16.
［24］Du H, Zeng X, Zhao M, et al. Efficient targeted mutagenesis in soybean by TALENs and CRISPR/Cas9［J］. Journal of Biotechnology, 2016, 217: 90-97.
［25］Kanazashi Y, Hirose A, Takahashi I, et al. Simultaneous site-directed mutagenesis of duplicated loci in soybean using a single guide RNA［J］. Plant Cell Reports, 2018, 37（3）: 553-563.
［26］Song S, Hou W, Godo I, et al. Soybean seeds expressing feedback-insensitive cystathionine gamma- synthase exhibit a higher content of methionine［J］. Journal of Experimental Botany, 2013, 64（7）, 1917-1926.
［27］Cai Y, Chen L, Liu X, et al. CRISPR/Cas9-mediated genome editing in soybean hairy roots［J］. PLoS One, 2015, 10(8): e0136064.
［28］Tang F, Yang S, Liu J, et al. Rj4, a gene controlling nodulation specificity in soybeans, encodes a thaumatin-like protein but not the one previously reported［J］. Plant Physiology, 2016, 170(1): 26-32.
［29］Fang Y, Tyler B M. Efficient disruption and replacement of an effector gene in the oomycete P hytophthora sojae using CRISPR/Cas9［J］. Molecular Plant Pathology, 2016, 17(1): 127-139.
［30］Cai Y, Chen L, Liu X, et al. CRISPR/Cas9-mediated targeted mutagenesis of GmFT2a delays flowering time in soya bean［J］. Plant Biotechnology Journal, 2018, 16(1): 176-185.
［31］Schmutz J, Cannon S B, Schlueter J, et al. Genome sequence of the palaeopolyploid soybean［J］. Nature, 2010, 463（7278）: 178-183.
［32］Miyahara A, Hirani T A, Oakes M, et al. Soybean nodule autoregulation receptor kinase phosphorylates two kinase-associated protein phosphatasesin vitro［J］. Journal of Biological Chemistry, 2008, 283(37): 25381-25391.
［33］Liu L, Lei X L, Huang G Z, et al. Influences of mechanical sowing and transplanting on nitrogen accumulation，distribution and C/N of hybrid rice cultivars［J］. Journal of Plant Nutrition and Fertilizer, 2014, 20(4): 831-844.

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备注/Memo

收稿日期：2019-02-25

基金项目：国家十三五重大研发计划（2016YFD0100700）。
第一作者简介：柏梦焱(1993-)，男，硕士，主要从事CRISPR-Cas9技术研发与分子育种研究。E-mail：461326458@qq.com。
通讯作者：关跃峰(1981-)，男，博士，教授，主要从事大豆碳氮代谢调控及基因编辑研究。E-mail：guan@fafu.edu.cn。

更新日期/Last Update: 2019-07-25